At 8 weeks after surgery, a segment of sciatic nerve comprising the tissue 1 mm away from the proximal and distal anastomotic stoma of CEANA was resected (mainly to observe whether regenerated axons passed through the anastomotic scar), fixed with 10% formaldehyde, embedded with paraffin, and successively sliced into sections (each section spanning the area 2 mm away from the proximal and distal anastomotic stoma). The transverse sections were routinely stained with hematoxylin-eosin, cut into semi-thin sections, stained with toluidine blue and observed under the optical microscope (Olympus, Tokyo, Japan). A 3 mm-long sciatic nerve segment was cut 3 mm away from distal anastomotic stoma of the sciatic nerve on the surgical side. An identical sciatic nerve segment from the control side was cut as a normal control. The specimen was rinsed, fixed with glutaraldehyde, embedded with epon 812, sliced into ultrathin cross-sections, counterstained with uranium-lead and observed under transmission electron microscope (CM120, Oxoid, Basingstoke, UK). Using the Image-Pro Plus image analysis system (Media Cybernetics, Bethesda, MD, USA), the total number of myelinated nerve fibers was counted and the thickness of myelin sheath was measured.
Cm120
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
The CM120 is a compact, high-performance benchtop centrifuge designed for general-purpose laboratory applications. It features an easy-to-use digital control panel, a wide range of speed and time settings, and a robust, quiet operation. The CM120 is suitable for a variety of sample types and volumes, making it a versatile tool for various laboratory workflows.
Automatically generated - may contain errors
Lab products found in correlation
Usc1000 2k 2k camera, by Ametek (2 mentions)
Tecnai g2 spirit, by Thermo Fisher Scientific (2 mentions)
Keenview, by Olympus (2 mentions)
Quemesa, by Olympus (2 mentions)
Image pro plus image analysis system, by Media Cybernetics (1 mentions)
Optical microscope, by Olympus (1 mentions)
Spectramax m5, by Molecular Devices (1 mentions)
Talos 200c, by Thermo Fisher Scientific (1 mentions)
Oxford ct 3500 cryo holder, by Philips (1 mentions)
Multiscan 791, by Philips (1 mentions)
Cm 120, by Ametek (1 mentions)
R1 2 1, by Quantifoil (1 mentions)
Cryo box, by Zeiss (1 mentions)
Gatan 626 dh, by Ametek (1 mentions)
Dmi6000b microscope, by Leica (1 mentions)
Temcam f416 cmos, by TVIPS (1 mentions)
Ct3500, by Ametek (1 mentions)
Us1000, by Ametek (1 mentions)
Multiscan 791, by Ametek (1 mentions)
Libra 120, by Zeiss (1 mentions)
17 protocols using cm120
1
Sciatic Nerve Regeneration Assessment
2
Triceps Surae Wet Weight and Sciatic Nerve Regeneration
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At 8 weeks after repair, the bilateral triceps surae were harvested and weighed using an electronic balance (type HZT A1000, Shanghai Jia Zhan Instrumentation Equipment, Shanghai, China) after surface blood clot was removed using gauze. Weight was calculated using the following formula: triceps wet weight recovery rate = (triceps wet weight at surgical side/triceps wet weight at control side) × 100%. A 7-mm sciatic nerve was cut at the inferior border of the piriformis muscle of the operated side, 3 mm lateral to the distal anastomosis. The specimen was rinsed, fixed with glutaraldehyde, and embedded using epoxy 812. Then the specimen was sliced into ultrathin cross-sections and counterstained with uranium-lead. Nerve regeneration was observed under transmission electron microscopy (CM120, Oxoid, Wade, UK). The myelin sheath thickness was measured using Image Pro Plus 5.0 system (Bethesda, Maryland, USA). Semithin sections of sciatic nerve specimens were stained with toluidine blue and observed under transmission electron microscopy (JEM-1200EX, Japanese Electronics Company, Tokyo, Japan), to calculate the total number of myelinated nerve fibers and the myelin thickness.
3
Negative Staining Transmission Electron Microscopy
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Samples (3.5 μl) were applied on carbon-coated and glow-discharged 300 mesh copper grids (SPI) and adsorbed for 1 min. The samples were then washed in two drops of 50 μl deionized water and stained with 50 μl of 1.5% uranyl acetate for 30 s. Negatively stained samples were visualized by TEM: JEOL 1230 operating at 80 kV and Philips CM120 or FEI Talos L120 operating at 120 kV. Digital images were recorded with Gatan Orios 4k-pixel CCD camera and Digital Micrograph software, Olympus CCD SIS Cantega G2 2k-pixel CCD camera using iTEM software, or FEI Ceta 16k-pixel CMOS camera and TIA software. Image brightness and contrast were adjusted using Adobe Photoshop CC software.
4
Hfq Peptide Filament Imaging via TEM
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One droplet of Hfq peptide filaments was applied to glow-discharged 300 mesh carbon-coated copper grids for 1 min, washed with water, stained with 2% uranyl acetate (w/v) for 1 min, and dried under dark conditions. Samples were observed using a FEI CM120 transmission electron microscope at an accelerating voltage of 120 kV under TEM low-dose mode. TEM images were recorded using a Gatan USC1000 2k × 2k camera.
5
Transmission Electron Microscopy Sample Preparation
6
Characterization and Degradation of Hollow Nanoparticles
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Size and Zeta (ζ) potentials were measured using a Nano-ZS90 Zetasizer equipment (Malvern, Westborough, MA, USA). Transmission electron microscopy (TEM, UC Davis School of Medicine, FEI CM120, Hillsboro, OR, USA) was used to investigate the nanoparticles structure and morphological aspect. First, a 7 μL droplet of MilliQ water containing suspended nanoparticles were placed in discharged TEM grids for 10 min. Then, samples were stained with uranyl acetate, dried, and imaged at room temperature. Confirmation of nanoparticle degradation was performed using hollow nanoparticles labeled with 1 mol% rhodamine B isothiocyanate. Degradation studies were performed in PBS pH 7.4 and PBS pH 3.5. The absorbance of the hollow nanoparticles dispersed at a final concentration of 1 mg/mL was monitored over a period of 7 days using a Spectramax M5 plate reader (Molecular Devices, Sunnyvale, CA, USA) at 544 nm.
7
Cryo-TEM Imaging of Nanoparticle Samples
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Samples were prepared to be at a mass percentage of 1% particles in PBS and the Cryo-TEM specimens were prepared in a controlled environment vitrification system (CEVS). Cryogenic transmission electron microscopy (cryo-TEM) imaging was performed either by a Phillips CM120 or a FEI Talos 200 C, FEG-equipped cryo-dedicated high-resolution transmission electron microscope (TEM and STEM), operated at an accelerating voltage of 120 kV. Specimens were transferred into an Oxford CT-3500 cryo-holder (Philips) or a Gatan 626DH (FEI) cryo-holder, and equilibrated below −178 °C. Specimens were examined using a low-dose imaging procedure to minimize electron-beam radiation damage. Images were recorded digitally by a Gatan Multiscan 791 cooled CCD camera (Philips CM 120), or a Gatan US 1000 high-resolution CCD camera (Tecnai T12 G2), using DigitalMicrograph software.
8
Cryo-TEM Imaging of Proteorhodopsin
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Cryo-TEM was performed as described earlier (Rhiel et al. 2013 (link)). Briefly, a small droplet (5 μl) of the proteorhodopsin fraction, i.e., band B3 was placed on a copper grid covered by a holey carbon film (Quantifoil R 1.2/1.3, pore size 1.2 μm, 400 mesh; Quantifoil Micro Tools, Jena, Germany). Excess liquid was blotted for 3 s between two strips of filter paper. Subsequently, the sample was rapidly plunged into liquid ethane (cooled to about − 180 °C with liquid nitrogen) in a Cryobox (Zeiss, Oberkochen, Germany). The frozen specimen was transferred with a cryo-holder (Gatan 626-DH, Gatan, Pleasanton, USA) into a precooled cryo-transmission electron microscope (CM 120, FEI, Eindhoven, Netherlands) operated at 120 kV and viewed under low dose conditions. Cryo-TEM images were recorded with a 2 K CMOS Camera (F216, software EMMENU V4.0; camera and software TVIPS, Munich, Germany).
9
Ultrastructural Characterization of Nanoparticles
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Cells were fixed using 1.6% (v/v) glutaraldehyde in phosphate buffer 0.1 M at pH 7.4 overnight at 4°C. They were postfixed using 1% (v/v) osmium tetroxide for 90 minutes and dehydrated in ascending series of ethanol dilutions. They were then treated with propylene oxide, impregnated in ascending dilutions of resin in propylene oxide, left in pure resin overnight (Epon; Inland Europe), embedded, and polymerized at 60°C for 48 hours. Ultrathin sections (70 nm) performed on an ultramicrotome (RMC, powertome PC) were collected on butvar-coated single-slot copper grids and were stained with 2% (v/v) uranyl acetate for 30 minutes and with lead citrate for 2 minutes. For NP characterization, solution at 40 μg/mL was deposited on a carbon-coated grid previously submitted to a glow discharge (Elmo, Cordouan Technologies). Grids were examined by transmission electron microscopy (TEM; CM120; FEI), and the images were acquired using a digital 2×2 k Gatan camera.
10
Quantifying Nanoparticle Uptake in Cells
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Cells were seeded (3 × 104 cells per glass bottom gridded dish, MatTek Corporation) in 2 mL of the appropriate growth medium and were grown for one day to reach 40% confluence. MPIO (0.3 mM Fe) or ScreenMAG (3.2 mM Fe) were incubated on cells using complete medium or free-serum medium, respectively (see Supplementary Data S5 A for more explanation). After 4 h of incubation, cells were washed with PBS and fixed with 4% PFA in PBS for 2 h at room temperature. Cells were observed in fluorescence microscopy using a Leica DMI6000B microscope equipped with a CCD camera. After washes in milliQ water, cells were post-fixed using 1% (v/v) osmium tetroxide in PBS for 1 h. Cells were embedded in Epon for ultramicrotomy. Ultrathin sections were stained with 2% uranyl acetate and lead citrate before examination using a 120 kV CM120 (FEI) transmission electron microscope.
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